Summary

An amputated cricket leg regenerates all missing parts with normal size and
shape, indicating that regenerating blastemal cells are aware of both their
position and the normal size of the leg. However, the molecular mechanisms
regulating this process remain elusive. Here, we use a cricket model to show
that the Dachsous/Fat (Ds/Ft) signalling pathway is essential for leg
regeneration. We found that knockdown of ft or ds
transcripts by regeneration-dependent RNA interference (rdRNAi) suppressed
proliferation of the regenerating cells along the proximodistal (PD) axis
concomitantly with remodelling of the pre-existing stump, making the
regenerated legs shorter than normal. By contrast, knockdown of the
expanded (ex) or Merlin (Mer) transcripts
induced over-proliferation of the regenerating cells, making the regenerated
legs longer. These results are consistent with those obtained using rdRNAi
during intercalary regeneration induced by leg transplantation. We present a
model to explain our results in which the steepness of the Ds/Ft gradient
controls growth along the PD axis of the regenerating leg.

INTRODUCTION

Regeneration depends on the recognition of tissue loss and the subsequent
restoration of the relevant structure. Many insights into the mechanisms
underlying such regeneration processes have been obtained from studies on limb
regeneration in urodeles (for reviews, see
Nye et al., 2003;
Brockes and Kumar, 2008) and
hemimetabolous insects (for a review, see
Nakamura et al., 2008a).
However, numerous questions remain, including how blastemal cells know their
positional identities, how they detect positional disparity along the
proximodistal (PD) axis, how they accurately restore the leg to its normal
size, and what factors govern blastemal cell proliferation in the correct
proportions. To address these questions, we have developed an experimental
system using the two-spotted cricket Gryllus bimaculatus
(Mito et al., 2002;
Nakamura et al., 2007;
Nakamura et al., 2008a;
Nakamura et al., 2008b;
Mito and Noji, 2009). Using
this system, we have studied the molecular biological basis of a number of
phenomena that were originally identified in a series of excellent classical
studies on insect leg regeneration (Bohn,
1965; French et al.,
1976; Meinhardt,
1982). The greatest advantage to our cricket system is that it
allows regeneration-dependent RNAi (rdRNAi) to be used for loss-of-function
analyses. Regeneration-dependent RNAi is a type of RNAi that occurs
specifically after leg amputation in cricket nymphs that have been injected
with double-stranded RNA (dsRNA) for a target gene
(Nakamura et al., 2008a). In
this system, when the metathoracic (T3) tibia of the third-instar nymph
(Fig. 1A) is amputated
(Fig. 1B), it takes
approximately 40 days (six ecdyses) to restore the adult leg. The process
begins with the covering of the amputated region by newly formed cuticle. A
ligand of Epidermal growth factor receptor (Gb'Egfr) is then induced
by Decapentaplegic (Gb'Dpp) and Wingless (Gb'Wg) in a
blastema composed of epithelial stem cells, which begins to undergo rapid
proliferation to restore the lost portion in the fourth instar
(Fig. 1B)
(Mito et al., 2002;
Nakamura et al., 2008b). In
the fifth instar, the tibiae, tibial spurs, tarsi and tarsal claws are
restored in miniature (Fig.
1B). In the seventh instar, the amputated legs restore the missing
portion to regain a nearly normal appearance
(Fig. 1B). As no leg
regeneration was observed after amputation in the case of rdRNAi against
Gb'armadillo (Gb'arm), the canonical Wnt pathway should be
involved in the initiation of the regeneration
(Nakamura et al., 2007).

To identify other genes involved in leg regeneration, we chose candidates
that have been implicated in Drosophila appendage PD patterning, and
examined their functions using the rdRNAi method. We were particularly
interested in molecules involved in the Dachsous/Fat (Ds/Ft) signalling
pathway, which has been extensively studied (for a review, see
Reddy and Irvine, 2008). Based
on these Drosophila data, we selected a set of 23 genes in the Ds/Ft
signalling pathway, as listed in Table
1 and shown in Fig.
1C (Reddy and Irvine,
2008). To determine whether these candidate Drosophila
genes are involved in cricket leg regeneration, we cloned cDNA fragments of
their Gryllus homologues (unbroken lines in
Fig. 1C). We examined the
effect of rdRNAi against each of the 23 target genes on regenerating legs, and
found at least 15 genes to be involved in leg regeneration, indicated in
yellow in Fig. 1C and listed in
Table 1. In this report, we
focus mainly on the functions of the Gryllus homologue of ft
(Gb'ft), Gb'ds, Gb'four-jointed (fj),
Gb'dachs (d), Gb'expanded (ex), or
Gb'Merlin (Mer). Others will be published elsewhere. In
brief, Ft is a large protocadherin that is evolutionarily conserved from
Drosophila to mammals (Reddy and
Irvine, 2008). In Drosophila, the protocadherins Ft and
Ds regulate planar cell polarity (PCP), PD patterning and cell proliferation
(Mahoney et al., 1991;
Clark et al., 1995;
Adler et al., 1998;
Yang et al., 2002), whereas
Fj, encoding a Golgi kinase, regulates Ft signalling by phosphorylating Ft and
Ds (Ma et al., 2003;
Strutt et al., 2004;
Ishikawa et al., 2008). D is
an unconventional myosin, acting as a crucial downstream component of the
Ds/Ft signalling pathway that influences growth, cell affinity and gene
expression during development (Mao et al.,
2006). Both ex and Mer encode cytoplasmic
proteins belonging to the Band 4.1 superfamily that are proposed to function
together in a redundant manner upstream of Hippo/Warts (Hpo/Wts) signalling to
regulate contact inhibition of cell proliferation
(Boedigheimer and Laughon,
1993; McCartney et al.,
2000; Hamaratoglu et al.,
2006). Our experimental results indicated that leg size and shape
are regulated through the Ds/Ft signalling pathway, including Ex/Mer, during
regeneration. These insights provide cues to understand molecular mechanisms
underlying a process of regeneration that was first described a century
ago.

MATERIALS AND METHODS

Animals

Cloning of the Gryllus homologues of
Ds/Ft-signalling-related genes

Gryllus genes homologous to the Drosophila
Ds/Ft-signalling-related genes were cloned by a degenerate PCR method or by
PCR using gene-specific primers in a reaction mixture containing LA-taq or
Ex-taq in a GC buffer (TaKaRa, Kyoto), as described previously
(Nakamura et al., 2007).
Degenerate primers were designed using the CODEHOP algorithm
(http://bioinformatics.weizmann.ac.il/blocks/codehop.html)
with conserved regions among insects and mammals, or were designed based on
conserved nucleic acid sequences among insect species. Gene-specific primers
were designed by probe search
(http://probe-search.ccr.tokushima-u.ac.jp/probe_search/index.html)
based on the nucleotide sequence of expressed sequence tag (EST) and
high-throughput cDNA (HTC) data on DNA Databank of Japan. The primer sequences
are listed in Table 2. The
sequences reported here were deposited in GenBank. The accession number of
each gene is listed in Table
1.

Sequences of primers and template cDNAs for cloning of Gryllus
homologous genes

Regeneration-dependent RNAi

Preparation of dsRNA and rdRNAi was as previously described
(Nakamura et al., 2007;
Nakamura et al., 2008b).
Briefly, dsRNAs were synthesized using the MEGAScript Kit (Ambion) and
adjusted to 20 μM. After injection of dsRNA into the abdomen of
third-instar nymphs, their tibiae were amputated between the second and third
spines. Because there are three spines in the tibia, the first being the most
distal, 30% of the distal portion of the tibia was removed by the amputation,
unless otherwise stated. As a negative control, we injected dsRNA for an
exogenous gene, DsRed2, which encodes a red fluorescent protein in
the nymph. We performed RNAi experiments with more than 20 nymphs twice for
each gene (Nakamura et al.,
2008b). Expressivity of phenotypes caused by nymphal RNAi was more
than 90%, unless otherwise noted. For dual RNAi experiments, a mixture of
dsRNAs for the two target genes was used. In the mixture, the final
concentration of each dsRNA was adjusted to 10 μM. In a control experiment
for the dual RNAi procedure, the dsRNAs were replaced by injection buffers
alone.

Nymph leg transplantation

Transplantation experiments for normal and reverse intercalary regeneration
and supernumerary leg formation were performed as described previously
(Mito et al., 2002;
Nakamura et al., 2007).
Briefly, we collected nymphs one day after moulting from the second to the
third instar. For transplantation experiments, we used the amputated
metathoracic leg stump as the host and the amputated mesothoracic distal
portion as the graft. To connect the legs, the mesothoracic graft was inserted
into the metathoracic leg stump of the same animal as used for graft
preparation under ice-chilled conditions and left for 1 hour. We tried to
transplant between control and RNAi legs. However, when a control graft was
transplanted to an RNAi host, RNAi was induced in the graft. Conversely, when
an RNAi graft was transplanted to a control host, the graft became normal.
Thus, we cannot show results for a case that one half of the regenerate is
treated with RNAi.

Cell proliferation assay

Cell proliferation assays were carried out using the Click-iT EdU Alexa
Fluor 488 Imaging Kit (Invitrogen) (Salic
and Mitchison, 2008). 5-Ethynyl-2′-deoxyuridine (EdU) was
injected into the abdomen of cricket nymphs at the appropriate stages after
amputation. Regenerating legs were fixed 4 hours after EdU injection.
EdU-incorporating cells were detected according the manufacturer's
instructions. Propidium iodide (PI) was used for nuclear staining.

Regenerating legs of Gryllus bimaculatus and the
Drosophila Ds/Ft signalling pathway. (A) Cricket nymph at
third instar. Mesothoracic and metathoracic legs are indicated by T2 and T3,
respectively. (B) Typical processes of cricket leg regeneration.
(C) Subcellular localization of the Ds/Ft signalling factors and their
network in Drosophila. The Gryllus homologues we cloned are
indicated by unbroken lines, with others shown as dashed lines. Yellow
indicates factors with RNAi phenotypes that were observed during
Gryllus leg regeneration.

Quantitative-PCR (q-PCR)

Total RNA was extracted from the regenerating tibiae of control, Gb'ft,
Gb'ds or Gb'hpo rdRNAi nymphs at the third or sixth instar, 2
days post-amputation (dpa), using the RNAqueous-Micro Kit (Ambion). mRNA (1μ
g) was reverse transcribed to cDNA using the SuperScript First-Strand
Synthesis System (Invitrogen) according to the manufacturer's instructions.
q-PCR was performed with the ABI 7900 Real-Time PCR System (Applied
Biosystems) as described previously
(Nakamura et al., 2008b). The
sequences of the q-PCR primers are as follows (forward and reverse, 5′
to 3′): ds(IC), CGGATGATAAACGAAGGTCCTCTA and
GGTTGTCATCATCTGCATCATCTC; fj, TCGTGTTCGACTACCTCACG and
TTCGTTGTCGAAGAAGACCA; ft(IC), TGCACCACCTCAACCACCT and
TCGTCGAGCTGTGGTTGTCC; hpo, AAAAGGAACACAGTCATAGGCACA and
AATGTCAGCAACACAATCATACCC; β-actin, TTGACAATGGATCCGGAATGT and
AAAACTGCCCTGGGTGCAT.

RESULTS

To examine the developmental roles of Gb'ft, Gb'ds and
Gb'fj, we attempted to knock down their expression by parental,
embryonic and nymphal RNA interference (RNAi)
(Mito and Noji, 2009). No
significant effect was observed in the treated eggs or nymphs, indicating that
RNAi against those genes is not effective at these stages. By contrast, rdRNAi
was highly effective, reproducible and constant; with this method, we found at
least 15 genes (yellow in Fig.
1C) to have phenotypes implicating them in leg regeneration (see
Fig. S1 in the supplementary material). We divided the leg phenotypes obtained
by rdRNAi into five classes: (1) distal enlargement; (2) abnormal regeneration
along the PD axis (short or long); (3) abnormal regeneration along the
circumferential axis (thin or thick); (4) others; and (5) no phenotype
(Table 1). To denote, for
example, a nymph containing injected dsRNA for Gb'ft, we use the term
`Gb'ft RNAi nymph'. Details of nymph phenotypes after RNAi treatment
of genes not shown here will be published elsewhere.

To confirm that rdRNAi decreased the amount of Gb'ft mRNA in
regenerating tibial stumps, we performed q-PCR. We estimated the ratio of the
amount of Gb'ft mRNA at 2 dpa compared to 0 dpa (n=10). The
average ratio at 2 dpa was lowered to 0.30±0.02 (in triplicate,±
standard deviation unless otherwise noted) in regenerating
Gb'ft RNAi tibiae, indicating that rdRNAi had occurred (see Fig. S2A
in the supplementary material). We also estimated the duration of the rdRNAi
effect in the nymphal leg. When legs were amputated at the sixth instar after
injection of dsRNA at the third instar, the relative ratio of Gb'ft
mRNA (n=10) at 2 dpa was not lowered to 1.06±0.06 (see Fig.
S2A in the supplementary material), indicating that RNAi was no longer
effective at the sixth instar. To ensure that there had been no off-target
effect, we compared the phenotypes obtained with two dsRNAs corresponding to
different regions of the same gene. In the case of rdRNAi against
Gb'ft or Gb'ds, we observed a similar `short and thick'
phenotype (see Fig. S2C,D in the supplementary material) as well, indicating
that the phenotypes obtained by rdRNAi are not off-target effects. We also
confirmed the specificity by which mRNA was depleted by rdRNAi (see Fig. S2B
in the supplementary material): in regenerating Gb'ft RNAi tibiae
(n=10), the relative ratio of Gb'ft mRNA at 2 dpa was
reduced (0.40±0.06), whereas that of Gb'ds was slightly
increased (1.38±0.31). Similarly, in regenerating Gb'ds RNAi
tibiae (n=10), the ratio of Gb'ds mRNA was reduced
(0.37±0.12), whereas that of Gb'ft was slightly increased
(1.28±0.36). In regenerating Gb'fj RNAi tibiae (n=3),
expression of Gb'fj became undetectable by whole-mount in situ
hybridization (data not shown). These results suggest that the observed effect
of rdRNAi was genuine and specific to each target gene.

To observe the expression pattern of Gb'ft, Gb'ds, Gb'fj and
Gb'd during leg development and regeneration, we performed
whole-mount in situ hybridization. Gb'ft and Gb'ds were
intensely expressed in limb buds at early stages
(Fig. 2A,B), whereas the
expression of Gb'fj or Gb'd was weak
(Fig. 2B; see Fig. S3 in the
supplementary material). At later stages, the expression of Gb'ft was
localized to the proximal region of each leg segment, where it showed a PD
gradient (Fig. 2B), whereas
Gb'ds and Gb'd were expressed in the distal region of each
segment (Nakamura et al.,
2008a). Expression of Gb'fj was observed in the proximal
region of each segment (Fig.
2B). To determine whether Gb'ft or Gb'ds are
expressed during regeneration, we performed whole-mount in situ hybridization
with regenerating legs in which the cuticle had been removed. We observed that
Gb'ft was expressed in the proximal region of the tarsus primordium
at 5 dpa (Fig. 2C), represented
by blue in the illustration (Fig.
2C, right), whereas expression of Gb'ds was observed in
distal regions of the regenerating tibia and tarsus primordia (represented by
green, Fig. 2C, right). No
significant signal was observed in negative controls
(Fig. 2C). The expression
pattern of Gb'ft and Gb'ds in regenerating legs resembled
that normally observed in late-stage limb buds
(Fig. 2B), suggesting that
these patterns are similar in each leg segment. However, Gb'ex was
expressed in the proximal region of each segment, whereas expression of
Gb'Mer was ubiquitous in leg buds (data not shown). Based on their
expression patterns, we concluded that these genes are involved in pattern
formation of the limb during development and regeneration.

Expression pattern of Gb'ft, Gb'ds, Gb'fj and
Gb'd in embryos, and of Gb'ft and Gb'ds in
regenerating legs 5 days after amputation. (A) Expression patterns
of Gb'ft in embryo at stages 5, 7 and 9, obtained by whole-mount in
situ hybridization (WISH). Gb'ft was expressed in the distal region
of the prothoracic limb, the head and abdominal regions at stage 5. At stages
7 and 9, Gb'ft was expressed in the antenna, limb, cercus and every
abdominal segment. (B) Expression patterns of Gb'ft, Gb'ds,
Gb'fj and Gb'd in developing limbs at stages 5, 7 and 9,
obtained by WISH. Gb'ft was expressed in the distal region of the
limb bud at stage 5, in the proximal region at stage 7 and thereafter in each
limb segment with a PD gradient. Gb'ds was expressed in the distal
region of the limb bud at stage 5, the most distal portion of the limb at
stage 7, and in the distal region of each limb segment at stage 9.
Gb'fj was expressed in the proximal region of each limb segment
between stages 7 and 9. Gb'd was expressed in the distal region of
each limb segment between stages 5 and 9. The red dotted lines indicate
segment boundaries. (C) Expression pattern of Gb'ft and
Gb'ds in regenerating legs at 5 dpa, obtained by WISH. The red dotted
lines indicate the segment boundary between the tibial and tarsal primordia.
An arrow indicates the proximal expression of Gb'ft. An arrowhead and
arrow indicate distal expression of Gb'ds in the tibial and tarsal
primordia, respectively. As negative controls, we used the corresponding sense
RNA probes (bottom). In controls, no significant signal was detected. The
observed areas of intense staining represent nonspecific binding of dyes to
small cuticular structures and tracheal tubes. (Right) Schematic of the
Gb'ft and Gb'ds expression patterns. Scale bar, 100 μm.
Cl, claw; Ta, tarsus; Ti, tibia.

The Ds/Ft signalling pathway participates in the control of leg
segment size and shape

We observed that the length of regenerated adult tibiae changed in nymphs
treated with RNAi against Gb'd, Gb'ds, Gb'ex, Gb'ft and
Gb'Mer (see Fig. S1 in the supplementary material). Because
Gb'ft and Gb'ds had shown different expression patterns in
the tibia, we examined whether the short phenotype depended on location of the
amputation. When the Gb'ft or Gb'ds rdRNAi tibia was
amputated proximally, we found that the regenerated adult legs became very
short and thick, and contained distal structures, including spines and spurs
on the tibiae and three joints of the tarsi and claws, whereas the
contralateral legs appeared normal. A typical adult with a `short and thick'
regenerate obtained by rdRNAi against Gb'ds is shown in
Fig. 3B, alongside a control
adult (Fig. 3A) for comparison.
As the legs of Gb'ds rdRNAi crickets
(Fig. 3D) resembled those of
controls (Fig. 3C), but on a
reduced scale, the pre-existing host stump of amputated tibiae may be
repatterned as a whole in nymphs treated with RNAi against Gb'ft,
Gb'ds or Gb'd. However, the thickening might be due to a PCP
defect, because Ft and Ds are known as regulators of PCP (for a review, see
Lawrence et al., 2008). As we
observed no defect in leg surface structures (see Fig. S4 in the supplementary
material), if it is the case, it must be uncoupled from the PCP of the surface
structures.

We next determined effects of the location of amputation on regenerated
tibial phenotypes at the sixth instar after injection of target-gene dsRNA at
the third instar (Fig. 3E). In
control regenerations, the missing portion of the leg was restored regardless
of the position of amputation. In the Gb'ft RNAi nymphs, we found the
length of the regenerating tibia to depend linearly on the amputated position.
When tibiae were amputated at proximal (30%), middle (50%), or distal (70%)
positions, they continued to grow and formed short and thick adult legs. The
length of the regenerated tibiae became 40±2% (n=8) of that of
the control tibia for proximal amputation, 52±6% (n=6) for
middle amputation and 74±14% (n=4) for distal amputation.
Thus, tibiae with RNAi against Gb'ft were shortened to a degree that
was in proportion to the position of amputation
(Fig. 3E). In Gb'ds or
Gb'd RNAi nymphs, the length of the regenerated tibiae was also
shorter in correspondence with the amputated positions
(Fig. 3E). However, no
significant change was observed in the length of the tibia of Gb'fj
RNAi nymphs (Fig. 3E). These
results imply that Gb'ft, Gb'ds and Gb'd appear to regulate
the size and shape of the leg segments during regeneration.

Effect of rdRNAi against Gb'ft, Gb'ds, Gb'fj,
Gb'd, Gb'ex and Gb'Mer on leg size, proliferation
of regenerating cells, and distal enlargement phenotypes. (A) A
control adult cricket with a normally regenerated leg. An arrow indicates the
site of amputation position. (B) A Gb'ds RNAi adult with a
short regenerated T3 leg (middle amputated position). Its short leg is shown
in D. (C,D) Control and Gb'ds rdRNAi legs at higher
magnification. Arrows indicate spurs of the tibia and tarsus, and arrowheads
indicate tibial spines. (E) Effects of rdRNAi on the size of
regenerating legs at sixth instar in control, Gb'ft, Gb'ds, Gb'fj, Gb'd,
Gb'ex and Gb'Mer RNAi nymphs. These regenerating legs were
amputated at distal, middle or proximal positions at the third instar. Sites
of amputation are indicated by red lines (top). (F) Effect of rdRNAi on
the proliferation of regenerating cells. PI staining (red) and EdU
incorporation (green) of regenerating cells in control, Gb'ft, or
Gb'ds RNAi nymphs at 2 dpa. Merged signals appear yellow. The upper
and lower panels show confocal z-stack images and sections of the
distal regions, respectively. Distal is to the right. (G) Effect of
single or dual RNAi on distal enlargement phenotypes at the fourth instar.
Asterisks indicate the enlargement phenotype. (H) Effects of single or
dual RNAi against Gb'ft and Gb'app, on regeneration of adult
legs.

In contrast to Gb'ft and Gb'ds, in the Gb'ex
RNAi nymphs, regenerated tibiae became longer by 25±4% (n=5)
than normally regenerated tibiae after distal amputation
(Fig. 3E). A similar phenotype
was observed even in proximal amputation in the Gb'ex RNAi nymphs
(Fig. 3E). In Gb'Mer
RNAi nymphs, the average length of regenerated tibiae exceeded that of
normally regenerated tibiae by 22±7% (n=8) after distal
amputation (Fig. 3E). This
phenotype was not observed in proximal or middle amputations
(Fig. 3E), which may indicate
redundancy between Gb'Mer and Gb'ex. These results suggested
that Gb'ex/Gb'Mer transcripts can influence determination of
leg size by inhibiting over-proliferation along the PD axis during
regeneration (see Fig. S5 in the supplementary material).

Proliferation of regenerating leg cells is regulated through the
Ds/Ft/Wts pathway

The short and thick phenotypes of legs treated with rdRNAi against
Gb'ft or Gb'ds appear to be due to abnormal regulation of
blastemal cell proliferation. Thus, we next examined the effect of
Gb'ft or Gb'ds rdRNAi on the proliferation of blastemal
cells at 2 dpa in normal regeneration, wherein blastemal cells begin to
rapidly proliferate so as to restore the lost portion of the leg. We performed
EdU incorporation assays (Salic and
Mitchison, 2008) in Gb'ft, Gb'ds and Gb'warts
(wts) RNAi nymphs. As shown in
Fig. 3F, EdU-positive cells
were localized to the epithelial cell layer underlying the wound surface. At 2
dpa, the number of positive cells in distal longitudinal sections of
Gb'ft, Gb'ds and Gb'wts RNAi regenerating legs was increased
in comparison with the corresponding control legs
(Fig. 3F). When calculated as
the ratio of the number of positive cells to the total number of cells in the
distal longitudinal section (average length 0.22±0.01 mm and width
0.18±0.01 mm, n=18) the value was 0.22±0.03
(n=5) for control legs, 0.50±0.11 (n=4) for
Gb'ft-RNAi, 0.48±0.19 (n=4) for Gb'ds-RNAi
and 0.47±0.18 (n=5) for Gb'wts- RNAi nymphs. These
data indicate that the proliferation of blastemal cells was enhanced in
Gb'ft, Gb'ds or Gb'wts rdRNAi nymphs.

To examine epistasis in the Ds/Ft signalling cascades, we analyzed the
phenotypes of dual RNAi knockdown of related genes, accomplished by
simultaneous injection of two different dsRNAs. The enlarged phenotype of the
Gb'ft or Gb'ds RNAi nymph was suppressed by dual rdRNAi
against Gb'd (Fig.
3G), consistent with a previous epistatic test in
Drosophila (Cho and Irvine,
2004; Cho et al.,
2006). The enlarged phenotype of Gb'wts RNAi nymphs was
also suppressed by dual RNAi against Gb'd, which implies that
Gb'D functions downstream of Gb'Wts in leg regeneration (see
Discussion). By contrast, we did not observe any phenotype in the
Gb'hippo (hpo), Gb'salvador (sav) or
Gb'Mob as tumor suppressor (mats) RNAi nymphs, or even in
triple knockdown nymphs, although relative amount of Gb'hpo
transcript in Gb'hpo RNAi tibiae was reduced to be 0.28±0.04
(n=10) (see Fig. S2E in the supplementary material). Both `distal
enlarged' and `short and thick' phenotypes of Gb'ft rdRNAi nymphs
were suppressed by dual RNAi with Gb'approximated (app)
(Fig. 3G,H), consistent with a
previous epistatic test in Drosophila
(Matakatsu and Blair, 2008).
The enlargement phenotypes of Gb'ft, Gb'ds and Gb'wts RNAi
nymphs were suppressed dual RNAi against Gb'yorkie (yki) and
weakly by dual RNAi against Gb'scalloped (sd)
(Fig. 3G), which indicates that
a transcriptional complex of Gb'Yki and Gb'Sd functions
downstream. These results suggested that the proliferation of regenerating
cells is regulated by the Wts pathway within the Ds/Ft signalling pathway.

The Ds/Ft signalling pathway is involved in intercalary
regeneration

Classical experiments using insect legs have demonstrated that, when two
leg stumps with disparate positional values are placed next to one another,
intercalary regeneration occurs to restore the missing positional values
(Fig. 4A)
(Bohn, 1965;
French et al., 1976). In
intercalary regeneration, it is known that most of the regenerated cells are
derived from the donor or host with the more distal positional identity
(Fig. 4A)
(Bohn, 1976;
French et al., 1976),
suggesting that regenerating cells inhibit the proliferation of more proximal
cells at the junction, a phenomenon known as `distal preponderance'
(Brockes and Kumar, 2008). The
extent and origin of a regenerated portion can be determined by observation of
its surface morphology (Fig.
4A). In the case of reverse intercalation, the PD polarity of
regenerates can be determined by the orientation of bristles.

To further explore the role of Ds/Ft signalling factors in intercalary
regeneration, we carried out transplantation experiments by grafting an
amputated T2 piece to a T3 host in the same animal. In the Gb'ft or
Gb'ds RNAi nymphs, although host-graft jointed regions enlarged,
neither normal nor reverse intercalary regeneration to restore the missing
region was observed (Fig. 4B).
Intercalary regeneration was similarly absent in Gb'd RNAi nymphs.
Instead, their host-graft jointed regions became thinner than normal,
reminiscent of the phenotype of reverse intercalation observed in
Gb'arm RNAi nymphs (Nakamura et
al., 2007). In Gb'fj RNAi nymphs, intercalary
regeneration occurred normally in both grafting experiments. These results
indicate that Gb'ft, Gb'ds and Gb'd are essential for
intercalary regeneration. In the Gb'ex or Gb'Mer rdRNAi
nymphs, both normal and reverse intercalary regeneration took place (see Fig.
S6A in the supplementary material), but regenerated cells were derived from
both distal and proximal pieces shown by EdU incorporation assay (see Fig. S6B
in the supplementary material). These results support the possibility that
Gb'ex and Gb'Mer are involved in the directional
contact-dependent inhibition of proliferation leading to a proximal
re-specification.

Schematic of leg transplantation experiments to show re-specification of
lost distal positional values in the short tibial stump of the Gb'ft
RNAi regenerated leg and the experimental results. (A) Schematic of
leg transplantation experiments. (Top) In the control transplantation, a
proximally amputated normal graft is transplanted at the sixth instar to a
distally amputated regenerate as a host, obtained by proximal amputation at
the third instar. As the lost distal positional values have been restored in
the host regenerate, a reverse intercalary regeneration was observed as shown
in B. (Bottom) Transplantation of a proximally amputated normal graft to a
distally amputated Gb'ft rdRNAi short tibia (obtained by proximal
amputation at the third instar) as a host at the sixth instar (in which rdRNAi
is no longer effective). If the lost distal positional values were restored
even in the short tibia, a reverse intercalary regeneration should be
observed, as observed in the control. (B) Experimental results.
Reversed intercalation was observed in both control and Gb'ft RNAi
short tibia, indicating that restoration of positional values takes place in
the stump. In higher magnification images (right column), reverse-oriented
bristles (leftward arrows; normal orientation, rightward arrows) were observed
in both regenerates. g, normal graft; h, regenerated stump after proximal
amputation at the third instar.

To examine whether circumferential positional information is affected in
RNAi nymphs, we performed an additional transplantation experiment to induce
supernumerary legs (Mito et al.,
2002) (see Fig. S7A in the supplementary material). Supernumerary
legs were formed in Gb'ft, Gb'ds, Gb'd and Gb'fj RNAi nymphs
after three moults subsequent to the transplantation experiment (see Fig. S7B
in the supplementary material). These results suggested that circumferential
positional information was normal in these RNAi nymphs, and that their
regenerating cells retained the ability to proliferate. We also examined the
effect of rdRNAi against Gb'ex and Gb'Mer on the formation
of supernumerary legs. We observed normal supernumerary legs in Gb'ex
RNAi nymphs, but not in Gb'Mer RNAi nymphs (see Fig. S7B in the
supplementary material), suggesting that Gb'Mer, but not
Gb'ex, may be involved in the regulation of proliferation along the
circumferential axis.

In the short Gb'ft, Gb'ds and Gb'd RNAi legs, although
their tarsi, tarsal claws and decorative structures were small, they were
essentially restored (Fig.
3B,D,E). This may indicate that cells in the distal end of the
short tibia possess the most distal positional identity, possibly due to a
re-specification of positional value. To test this possibility, we performed
transplantation experiments (Fig.
5A). If re-specification would occur in the leg of the
Gb'ft RNAi nymphs, so as to allow the acquisition of the missing
distal positional values in the shortened tibia
(Fig. 5A), then it would be
expected that, when normal mesothoracic tibiae were amputated proximally and
grafted to distally amputated regenerating metathoracic tibial host at the
sixth instar, reverse intercalary regeneration would be observed. It should be
noted that RNAi is no longer effective in a regenerating host leg of
Gb'ft RNAi nymph at the sixth instar, when injected with dsRNA at the
third instar (see Fig. S2A in the supplementary material). We observed
reversed orientation of the surface bristles in the regenerates
(Fig. 5B), indicating that a
reversed intercalation took place at the graft-host junction. Thus, we
concluded that the positional values of the tibial stump cells were
re-specified in the short rdRNAi tibia during moulting.

DISCUSSION

Using an RNAi knockdown approach against 23 candidate genes, we identified
15 components of the Ds/Ft signalling pathway that are involved in cricket leg
regeneration (Fig. 1C;
Table 1). Based on additional
data from Gryllus and Drosophila
(Reddy and Irvine, 2008), we
propose a model signalling cascade for the regulation of leg regeneration by
the Ds/Ft signalling pathway (Fig.
6). As the main components of the Ds/Ft signalling pathways are
conserved in vertebrates (Reddy and
Irvine, 2008), this signalling cascade may also be involved in
vertebrate leg regeneration. In the remainder of this section we will discuss
how the Ds/Ft signalling pathway might be involved in leg regeneration.

The Ds/Ft signalling pathway is involved in leg regeneration

The most typical phenotypes in the present data were the short and thick
legs induced by rdRNAi against Gb'ft or Gb'ds
(Fig. 3C,D,E). It is known that
the size of each leg segment normally scales with overall body size, a
phenomenon known as allometry (Shingleton
et al., 2007). Surprisingly, the size of the regenerated legs in
the phenotypes we observed did not scale with overall body size
(Fig. 3B). Furthermore, the
size of the regenerated legs depended upon the site of tibial amputation
(Fig. 3D,E). It is noteworthy
that, although the expression patterns of Gb'ft and Gb'ds
were different (Fig. 2C), their
short and thick phenotypes were similar. This is consistent with the fact that
Drosophila mutant phenotypes of both ft and ds in
adult legs are short and thick, despite the fact that ft and
ds have distinct expression patterns in Drosophila imaginal
discs (Garoia et al., 2000;
Ma et al., 2003). Thus, we
conclude that the activity of Ds/Ft signalling regulates leg segment size and
shape during regeneration. Furthermore, we showed that the Ds/Ft signalling
pathway may regulate leg size during regeneration through the Hpo signalling
pathway (Fig. 6A). This is also
supported by the fact that the Hpo signalling pathway is involved in an
intrinsic mechanism that restricts organ size
(Edgar, 2006;
Dong et al., 2007;
Pan, 2007;
Yin and Pan, 2007;
Lawrence et al., 2008) and
that the Ds/Ft signalling system defines a cell-to-cell signalling mechanism
that regulates the Hpo pathway, thereby contributing to the control of organ
size (Cho et al., 2006;
Rogulja et al., 2008;
Willecke et al., 2008).

A plausible Ds/Ft signalling pathway model for leg regeneration.
(A) After amputation, a ligand (Egf) of Egfr may be induced by Dpp/Wg
via Arm in the blastemal cells, establishing the most distal positional value
at the amputated surface (Mito et al.,
2002; Nakamura et al.,
2008b). The positional disparity is linked to regulation of
cellular proliferation through the Ds/Ft. D mediates the signal for
regeneration along the PD axis, probably through the Hpo/Wts pathway
(Fig. 1C;
Table 1). The Ds/Ft also
regulates proliferation along the circumferential axis. The Ex/Mer is involved
in contact-dependent inhibition of proliferation in the stump. Signalling
factors that may activate the Ex/Mer are unidentified. (B) A plausible
genetic cascade of Ds/Ft signalling factors for proliferation of leg blastemal
cells. Blastemal cell proliferation is regulated by the activity of Ds/Ft
through App and Hpo/Wts signalling factors, as revealed by dual rdRNAi. Dotted
lines indicate potential interactions derived from the Drosophila
data (Reddy and Irvine,
2008).

Meinhardt pointed out that two processes operate during leg regeneration.
One, which operates during the restoration of distal structures, is instructed
by a morphogen epidermal growth factor (Egf), which is itself induced by two
morphogens, Dpp and Wg, at the amputated surface
(Meinhardt, 1982;
Mito et al., 2002;
Nakamura et al., 2008a). The
other, operating in intercalary regeneration, is directly controlled by
neighbouring cells at the junction between host and graft, but not by
long-range morphogens (Meinhardt,
2007; Nakamura et al.,
2008a). It is likely that the Ds/Ft signalling pathway
participates in both mechanisms, because rdRNAi against Gb'ft or
Gb'ds affected leg regeneration after either distal amputation or
intercalary transplantation. In the case of distal amputation, as the
Gryllus tarsi and claws were not restored after tibial amputation in
the Gb'Egfr rdRNAi nymphs
(Nakamura et al., 2008a), we
have speculated that Gb'Egf functions as a morphogen in the leg
regeneration, as found in Drosophila leg imaginal discs
(Campbell, 2002;
Galindo et al., 2002).
Recently, Rogulja et al. (Rogulja et al.,
2008) demonstrated in the Drosophila wing disc that the
Fat signalling pathway links the morphogen-mediated establishment of gradients
of positional values across developing organs to the regulation of organ
growth. Thus, we speculate that the Ds/Ft system links the Egf-mediated
establishment of gradients of positional values across regenerating blastemal
cells to the regulation of regenerate growth.

As Gb'd rdRNAi legs exhibited the short-leg phenotypes, but not
thick ones (Fig. 3E), and
Gb'd is epistatic of Gb'ft and Gb'ds, Gb'D may
mediate the components of Ds/Ft signalling controlling leg size
(Fig. 6A). The enlarged
phenotype of Gb'wts RNAi nymphs was suppressed by RNAi against
Gb'd in Gryllus, indicating that Gb'd is in the
downstream of Gb'wts (Fig.
6B). This result differs from Drosophila data (broken
line in Fig. 6B)
(Cho et al., 2006). A genetic
analysis is necessary to confirm the difference, because the epistatic allele
is not null in RNAi experiments. As the phenotype of rdRNAi treatment against
Gb'ds was weaker than that against Gb'ft, as reported in the
corresponding Drosophila mutants
(Matakatsu and Blair, 2006),
Gb'Ft may interact with factors that are as yet unidentified.
Although the effect of rdRNAi against Gb'fj on leg size was very
mild, we cannot exclude the possible involvement of Gb'fj in
allometric growth. The short phenotypes were observed in the Gb'sd
RNAi nymphal legs (see Fig. S1 in the supplementary material), so it remains a
possibility that Gb'sd is involved in allometric leg growth
(Fig. 6A). However, the
apparent contribution of the Hpo-Sav-Mats complex is as yet uncertain
(indicated by a broken line in Fig.
6B).

We demonstrated that regenerated legs of Gb'ex and Gb'Mer
RNAi adults become longer than normal control legs
(Fig. 3E; see Fig. S1 in the
supplementary material), and that Gb'ex and Gb'Mer regulate
cell proliferation induced by the presence of positional disparity (see Fig.
S6A,B in the supplementary material). These results suggest that
Gb'ex and Gb'Mer are also involved in allometric growth of
the leg segment (Fig. 6A). In
Drosophila, it was reported that Ex and Mer negatively regulate cell
growth and proliferation through the Hpo/Wts pathway
(Hamaratoglu et al., 2006;
Pellock et al., 2007). In
mammalian cells, Nf2 (merlin) is known to be a crucial regulator of
contact-dependent inhibition of proliferation
(Curto and McClatchey, 2008).
Thus, we conclude that activities of Ex and Mer may regulate contact-dependent
inhibition of proliferation via the Wts signalling pathway to restore the
proper leg segment size during regeneration
(Fig. 6A).

The Ds/Ft signalling pathway links positional and allometric
information to determine regenerated leg size and shape: interpretation
according to the Ds/Ft steepness model

A widely accepted model for leg regeneration is the intercalation model,
based on positional information (Wolpert,
1969; Wolpert,
1994; Nye et al.,
2003; Brockes and Kumar,
2008). This model is based on the intercalation of new structures
so as to re-establish continuity of positional values during regeneration.
However, on the basis of this model, it is difficult to explain the changes in
leg size that were observed in the present study. Thus, we need to extend it
to include the control of growth and tissue size during regeneration. Several
models have been proposed to explain how organ size is regulated
(Garcia-Bellido and Garcia-Bellido,
1998; Day and Lawrence,
2000; Lawrence,
2004). Recently, Lawrence, Struhl and Casal
(Lawrence et al., 2008)
proposed a model, which we refer to here as the Ds/Ft steepness model, to
explain the mechanisms underlying the determination of organ size and PCP,
including the Warts/Hippo pathway as the mechanism for controlling growth
(Rogulja et al., 2008;
Willecke et al., 2008) in the
previous model (Casal et al.,
2002; Lawrence et al.,
2004; Casal et al.,
2006). In their model, they hypothesized that: (1) the morphogens
responsible for the overall pattern of an organ establish and orient the Ds/Ft
system, which then forms a linear Ds/Ft gradient. The nature of the Ds/Ft
gradient is unknown, although the number of Ds/Ft trans heterodimers is the
key variable; (2) the steepness of the Ds/Ft gradient regulates Hpo target
expression and cell proliferation, and its direction provides information used
to establish the correct cellular polarity; (3) growth would be expected to
cease when the slope of the gradient declines below a certain threshold value;
and (4) the maximum and minimum limits of the system are conserved, while
recently divided cells take up intermediate scalar values from their
neighbours.

Schematics of the Ds/Ft steepness model for leg regeneration.
(A) Normal allometric growth: the positional values (PVs) are denoted
arbitrarily by the numbers 1 to 9, whereas the scalar values of the Ds/Ft
gradient are indicated by the orange shading in the Ds/Ft steepness model
(Lawrence et al., 2008). The
scalar value of the Ds/Ft gradient is minimum at the most distal value, PV=9.
The steepness of the gradient at each point, measured as a differential across
each cell, correlates with the size along the PD axis in the leg. Growth would
be predicted to cease when the slope of the gradient fell below a certain
threshold value. (B) Normal regeneration: after amputation at PV=3 in
the tibia (left side, the tibial stump is indicated by orange), blastemal
cells detect positional disparity (PVs, 3/9) through the Ds/Ft signalling
pathway, and then a steeply sloped Ds/Ft gradient is formed, which leads to
intercalary growth until the re-establishment of positional continuity
(yellow, PV=4-8), as epimorphic-like regeneration. The pre-existing stump
(orange) grows allometrically, retaining the original positional and
allometric information (PV=1-3). (C) No regeneration: the pre-existing
stump grows without restoring the missing portion, having the original
positional and allometric information (PVs=1-3). The phenotype was observed in
the Gb'arm rdRNAi leg (Nakamura
et al., 2007). (D) Morphallaxis-like regeneration in
Gb'ft, Gb'ds or Gb'd rdRNAi nymphs after proximal
amputation. No epimorphic-like regeneration takes place by suppression of
proliferation of blastemal cells along the PD axis, although remodelling of
the stump takes place as morphallaxis-like regeneration. The positional values
are re-established in relation to the new tibia-tarsus boundary, in which
information about the ultimate tibial size (allometric information) is lost.
The normal Ds/Ft gradient, indicated by a dotted line, would shift down with
the same slope so as to reset the positional value of the amputated surface to
the most distal positional value, or the minimum scalar value of the Ds/Ft
gradient. The short leg size induced by rdRNAi against Gb'ft is well
interpreted with this model (continues in Fig. S8 in the supplementary
material).

Using their model, we propose a modified Ds/Ft steepness model for leg
regeneration acting as follows. Our results indicate that nymphal leg
regeneration depends on two major processes
(Fig. 7B): proliferation and
differentiation of blastemal cells (yellow in
Fig. 7B) and growth of the
pre-existing stump (orange in Fig.
7B). In each of these processes, new positional identities are
specified in relation to new segment boundaries. According to the Ds/Ft
steepness model, in normal regeneration, a very steep gradient should be
formed in the regenerating blastema (Fig.
7B). The regenerate may grow so as to restore the normal
pre-existing steepness. Reassignment of positional identities after amputation
will correlate with a similar re-setting of the minimum Ds/Ft scalar value,
and the results are consistent with the steepness hypothesis.

Growth of the pre-existing stump is a normal component of leg growth, in
which the pre-existing stump cells proliferate according to some allometric
signals, which may be related to the maximum scalar value and a slope of the
gradient, keeping their original positional information. This was observed in
the truncated leg of Gb'arm rdRNAi adults
(Nakamura et al., 2007)
(Fig. 7C).

In the absence of the proliferation and differentiation of blastemal cells,
as observed in the Gb'ft rdRNAi leg, the minimum scalar value, which
is the most distal positional value, would be established at the site of
amputation, and the Ds/Ft gradient would be expected, in turn, to shift down
with the same slope as the pre-existing one (pink,
Fig. 7D). The Ds/Ft steepness
model provides an explanation for the observation that the final leg size
depends on the amputated position, if we assume that the gradient shifts down
with the same slope as that where cells at an amputated position have the
minimum scalar value (pink, Fig.
7D; see Fig. S8 in the supplementary material). Thus, the observed
re-specification of regeneration legs induced in the legs treated with rdRNAi
against Gb'ft or Gb'ds is as would be predicted by the Ds/Ft
steepness model. Thus, it is likely that the Ds/Ft gradient functions to link
positional and allometric information to the regulation of leg segment growth.
Furthermore, if we assume that the activity of Ex/Mer is related to a
threshold value of the slope of the gradient that determines when growth
ceases (see Fig. S8 in the supplementary material), we can interpret all
rdRNAi phenotypes in the present study consistently with the Ds/Ft steepness
model for regeneration (Fig. 7;
see Fig. S8 in the supplementary material).

Recently, da Silva et al. (da Silva et
al., 2002) found that, in newt leg regeneration, a cell surface
protein with a glycosylphosphatidylinositol anchor, Prod 1, is implicated in
the local cell-to-cell interactions mediating PD positional identity
(Brockes and Kumar, 2008). As
the Ds/Ft signalling pathway is conserved in vertebrates
(Reddy and Irvine, 2008), the
pathway should be involved in vertebrate leg regeneration, probably
interacting with the Prod 1 system.

Supplementary material

Footnotes

We thank K. D. Irvine, H. Meinhardt, P. A. Lawrence and two anonymous
reviewers for critical comments and valuable suggestions, and Y. Tanaka for
technical advices. This work was supported by a grant from the
Ministry of Education, Culture, Sports, Science and Technology
of Japan to T.M., H.O., and S.N. and by a grant from the
Knowledge Clusters Initiative Projects to T.B.,
H.O. and S.N.

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